The invention is in the general field of proteins involved in Alzheimer""s disease.
Various genes and gene products involved in the development of Alzheimer""s disease have been identified. Neuritic plaques characteristic of the disease are composed of xcex2-amyloid (Axcex2), which are oligopeptides of about 40-43 amino acids in length derived from the xcex2-amyloid precursor protein (xcex2APP). Mutations in the gene encoding xcex2APP are associated with some cases of familial Alzheimer""s disease. Other cases of familial Alzheimer""s disease have been associated with mutations in two other loci, presenilin-1 and presinilin-2.
The invention is based on the discovery of a heretofore undescribed protein, which has been named ALARM or xcex4-catenin, on the basis of its interaction with presenilin 1. ALARM shows a striking sequence similarity to members of the armadillo (arm)-plakoglobin-xcex2 catenin protein family. In addition, ALARM transcripts are confined almost exclusively to brain tissue.
In addition to the specific human ALARM sequences provided (or cross-referenced) herein, molecules relevant to the invention include fragments of those sequences and related polypeptides, non-peptide mimetics, and nucleic acid sequences. The invention also includes antibodies to ALARM polypeptides. These polypeptides, as well as nucleic acid encoding them, can be used for a variety of diagnostic and therapeutic applications.
In one aspect the invention features a substantially pure vertebrate ALARM polypeptide, e.g, an ALARM polypeptide from a mammal such as the human ALARM polypeptide shown in FIG. 1 (SEQ ID NO:2).
By xe2x80x9cproteinxe2x80x9d and xe2x80x9cpolypeptidexe2x80x9d is meant any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation).
Polypeptides include, but are not limited to: recombinant polypeptides, natural polypeptides, and synthetic polypeptides as well as polypeptides which are preproteins or proproteins.
One way to ascertain purity of a preparation is by per cent dry weight. Generally, useful preparations are at least 60% by weight (dry weight) the compound of interest, i.e., an ALARM polypeptide. Preferably the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight the compound of interest. Purity can be measured by any appropriate standard method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. A xe2x80x9cmature human ALARMxe2x80x9d is the amino acid sequence shown in FIG. 1 SEQ ID NO:2.
Polypeptides substantially identical to mature human ALARM may have an amino acid sequence which is at least 85%, preferably 90%, and most preferably 95% or even 99% identical to the amino acid sequence of the ALARM polypeptide of the FIG. 1. SEQ ID NO:2. When assessing sequence identity of polypeptides, the length of the reference polypeptide sequence will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably 35 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 110 nucleotides.
Sequence identity can be measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705).
In the case of polypeptide sequences which are less than 100% identical to a reference sequence, the non-identical positions are preferably, but not necessarily, conservative substitutions for the reference sequence. Conservative substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine.
Where a particular polypeptide is said to have a specific percent identity to a reference polypeptide of a defined length, the percent identity is relative to the reference peptide. Thus, a peptide that is 50% identical to a reference polypeptide that is 100 amino acids long can be a 50 amino acid polypeptide that is completely identical to a 50 amino acid long portion of the reference polypeptide. It might also be a 100 amino acid long polypeptide which is 50% identical to the reference polypeptide over its entire length. Of course, many other polypeptides will meet the same criteria.
Polypeptides corresponding to one or more domains of ALARM are also within the scope of the invention. Thus, also featured is a polypeptide including at least one antigenic determinant of ALARM, a polypeptide comprising at least one copy of the 42 amino acid arm repeat in the ALARM polypeptide, or a polypeptide comprising a xcex2APP binding domain of ALARM. Preferred polypeptides are those which are soluble under normal physiological conditions.
The polypeptides of the invention can be expressed fused to another polypeptide, e.g., a marker polypeptide or fusion partner. For example, the polypeptide can be fused to a hexa-histidine tag to facilitate purification of bacterially expressed protein or a hemagglutinin tag to facilitate purification of protein expressed in eukaryotic cells.
In another aspect, the invention also features a substantially pure polypeptide which includes a first portion and a second portion; the first portion includes an ALARM polypeptide and the said second portion includes a detectable marker. The first portion can be either a full-length form of ALARM or one or more domains thereof. The first portion is fused to an unrelated protein or polypeptide (i.e., a fusion partner) to create a fusion protein.
The invention also includes a pharmaceutical composition which includes an ALARM polypeptide.
In still another aspect the invention features a recombinant nucleic acid encoding an ALARM polypeptide. In one preferred embodiments the nucleic acid encodes a soluble ALARM polypeptide.
The invention also features isolated nucleic acids encoding polypeptides corresponding to one or more domains of ALARM or ALARM-related polypeptides discussed above. ALARM-encoding nucleotides can include the nucleic acids shown in FIG. 1 SEQ ID NO:1, e.g., nucleotides 366-2636 of FIG. 1 SEQ ID NO:1. Also encompassed within the invention are nucleic acid sequences that encode forms of ALARM in which sequences are altered or deleted.
By xe2x80x9cisolated nucleic acidxe2x80x9d is meant nucleic acid that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5xe2x80x2 end and one on the 3xe2x80x2 end) in the naturally occurring genome of the organism from which it is derived. Thus, a recombinant nucleic acid could include some or all of the 5xe2x80x2 non-coding (e.,g., promoter) sequences which are immediately contiguous to the coding sequence. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus, such as a retrovirus; or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.
Nucleic acid sequences substantially identical to human ALARM sequences have a nucleotide sequence which is at least 85%, preferably 90%, and most preferably 95% or even 99% identical to the amino acid sequence of the ALARM polypeptide of FIG. 1 SEQ ID NO:2. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 110 nucleotides.
Also within the invention are nucleic acids encoding hybrid proteins in which a portion of ALARM or a portion (e.g., one or more domains) thereof is fused to an unrelated protein or polypeptide (i.e., a fusion partner) to create a fusion protein.
The nucleic acid can be isolated either as a matter of purity or by including in it in DNA that is a non-naturally occurring molecule; for example, the DNA is not immediately contiguous with both of the sequences with which it is immediately contiguous (one on the 5xe2x80x2 end and one on the 3xe2x80x2 end) in the naturally occurring genome of the organism from which it is derived. Thus, a recombinant nucleic acid could include some or all of the 5xe2x80x2 non-coding (e.,g., promoter) sequences which are immediately contiguous to the coding sequence. Other examples are a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a CDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.
The nucleic acids of the invention include nucleic acids encoding ALARM polypeptides fused to a polypeptide which facilitates secretion, e.g., a secretory sequence. Such a fused protein is typically referred to as a preprotein. The secretory sequence can be removed by the host cell to form the mature protein. Also within the invention are nucleic acids that encode mature ALARM fused to a polypeptide sequence to produce an inactive preprotein. Preproteins can be converted into the active form of the protein by removal of the inactivating sequence.
The invention also encompasses nucleic acids that hybridize under stringent conditions to a nucleic acid encoding an ALARM polypeptide. xe2x80x9cStringent conditionsxe2x80x9d means hybridization at 50xc2x0 C. in Church buffer (7% SDS, 0.5% NaHPO4, 1 mM EDTA, 1% BSA) and washing at 50xc2x0 C. in 2xc3x97SSC. The hybridizing portion of the hybridizing nucleic acids are preferably 20, 30, 50, or 70 bases long. Preferably, the hybridizing portion of the hybridizing nucleic acid is 95% or even 98% identical to the sequence of a portion of a nucleic acid encoding an ALARM polypeptide. Hybridizing nucleic acids of the type described above can be used as a cloning probe, a primer (e.g., a PCR primer), or a diagnostic probe. Preferred hybridizing nucleic acids encode a polypeptide having some or all of the biological activities possessed by naturally-occurring ALARM. Hybridizing nucleic acids can be splice variants encoded by one of the ALARM genes described herein. Thus, they may encode a protein which is shorter or longer than the various forms of ALARM described herein. Hybridizing nucleic acids may also encode proteins which are related to ALARM (e.g, proteins encoded by genes which include a portion having a relatively high degree of identity to an ALARM gene described herein).
The term xe2x80x9cnucleic acidxe2x80x9d encompasses both RNA and DNA, including cDNA, genomic DNA, and synthetic (e.g., chemically synthesized) DNA. The nucleic acid may be double-stranded or single-stranded. Where single-stranded, the nucleic acid may be the sense strand or the antisense strand.
In yet another aspect, the invention features vectors which include a nucleic acid of the invention. In one preferred embodiment, the nucleic acid of the invention is properly positioned for expression.
By xe2x80x9cpositioned for expressionxe2x80x9d is meant that the selected DNA molecule is positioned adjacent to one or more sequence elements which direct transcription and/or translation of the sequence such that the sequence elements can control transcription and/or translation of the selected DNA (i.e., the selected DNA is operably associated with the sequence elements). Such operably associated elements can be used to facilitate the production of an ALARM polypeptide.
In a still further aspect, the invention features transformed cells harboring a nucleic acid encoding ALARM sequences discussed above.
By xe2x80x9ctransformed cellxe2x80x9d is meant a cell into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a DNA molecule encoding (as used herein) ALARM polypeptide.
The invention also features purified antibodies which specifically bind an ALARM protein or polypeptide.
By xe2x80x9cpurified antibodyxe2x80x9d is meant an antibody which is at least 60%, by dry weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by dry weight, antibody.
By xe2x80x9cspecifically bindsxe2x80x9d is meant an antibody that recognizes and binds to and forms a complex with, a particular antigen, e.g., ALARM polypeptide, but which does not substantially recognize and bind to other molecules in a sample, e.g., a biological sample, which naturally includes ALARM.
The invention also features a method of diagnosing in a mammal, e.g., a human subject, an increased likelihood of, inclination toward, or susceptibility to developing a disease, in which a mutant form of the ALARM protein is a causative agent. The same method is also used to diagnose the ability of a mammal, e.g., a human, to transmit to future generations a mutant form of a protein which is a causative agent of a disease. The method involves analyzing the DNA of the mammal to determine the presence or absence of a mutation in a gene for an ALARM protein, the presence of such a mutation indicating the increased likelihood. Preferably the DNA is analyzed by amplifying the DNA with, e.g., the polymerase chain reaction, and identifying mutations in the DNA by use of the single-strand conformation polymorphism (SSCP) technique, as used and described herein, or by direct DNA sequencing.
In another aspect, the invention includes a method of inhibiting expression of an ALARM gene comprising administering to a cell containing an ALARM transcript an anti-sense ALARM oligonucleotide.
The invention also includes a method of detecting presenilin 1 in a sample, e.g., a sample taken from a human, comprising contacting the sample with an ALARM polypeptide. The sample can be from, e.g., cerebrospinal fluid.
In another aspect, the invention includes a method of diagnosing in a human subject a disease in which a mutant form of a protein which interacts with ALARM is a causative agent. The method includes analyzing a sample of fluid from the human subject to determine the presence or absence of the ALARM-interacting protein.
The invention further includes a method of diagnosing in a human subject an increased likelihood of developing or transmitting to future generations a disease in which a mutant form of a human ALARM is a causative agent. The method includes analyzing the DNA of the subject to determine the presence or absence of a mutation in a gene for an ALARM protein, the presence of such a mutation indicating the increased likelihood of transmitting the disease. The method can include, e.g., amplifying the DNA of the subject, DNA sequencing, or identifying a single strand conformation polymerism.
The invention also includes a probe or primer comprising a substantially purified single-stranded oligonucleotide, e.g., a DNA oligonucleotide, wherein the oligonucleotide contains a region which is identical to the sequence of a six-nucleotide, single-stranded segment of a gene encoding a mutant form of a human ALARM, wherein the segment comprising part or all of the mutation.
In yet another aspect, the invention includes a method of detecting an ALARM-containing complex in a biological sample by contacting the sample with an ALARM protein or an ALARM antibody and determining whether the ALARM protein or antibody binds to a component of the sample.
In a further aspect, the invention includes a method of diagnosing altered levels, e.g., lower or altered levels, of presenilin 1 in a sample by contacting the sample with ALARM and determining whether the sample contains presenilin 1 that binds to ALARM.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed descriptions, and from the claims.